Upon UV radiation or ionizing radiation, DNA replication forks can be stalled by inter-strand and intra-strand cross links. Stalled replication forks trigger assembly of the cellular DNA damage response machinery, which requires that some enzyme components of the DNA replication complex switch their physical locations, interaction partners and functions. One such component is FEN1 nuclease. Our preliminary data indicate that upon UV radiation, FEN1 switches from being a flap endonuclease for RNA primer removal and interacting with PCNA, to being a gap-dependent endonuclease for resolution of stalled DNA replication forks and interacting with WRN. This critical switch is mediated by a change in FEN1's post-translational modification (PTM) profile. These PTMs can act as a 'molecular barcode' that directs different FEN1-mediated protein- protein interactions to allow a switch of FEN1's functions. Disruption of the normal program of these PTMs may lead to uncontrolled cell growth and cancer. In this competitive renewal application, our goal is to establish a comprehensive relationship among genetic alterations, functional deficiency, and pathological consequences, using FEN1 nuclease as a model protein and transgenic mice as a model system. During the previous funding cycle we identified FEN1 mutations in cancer cells that eliminate the structural elements responsible for FEN1's nuclease activities, protein/protein interactions and PTMs. We also established corresponding knock-in mouse models that mimic the point mutations identified in human cancer, which enabled us to initially define the molecular and cellular events of tumorigenesis. These mouse models and our initial studies have prompted new aims to investigate the molecular mechanisms of FEN1mutation-mediated cancer pathogenesis caused by elevated mutagenesis during Okazaki fragment maturation and aberrant PTMs. The Specific Aims include: 1) To determine how FEN1-mediated 5' editing of -segment eliminates mispairs in Okazaki fragments and contributes to cancer avoidance. 2) To determine the role of FEN1/WRN complex in maintaining the stability of tandem repeat sequences and cancer avoidance. 3) To determine how UV radiation-induced post-translational modifications mediate a switch of FEN1's role from RNA primer removal to resolution of stalled replication forks. Successful completion of the proposed studies will generate important new knowledge about the function and regulation of FEN1 in maintenance of genome stabilities and cancer avoidance. In addition, the results are anticipated to have a high potential impact, as they may suggest new avenues for cancer prevention and development of new, personalized radiation and other therapeutic regimens for this life-threatening disease.